Stimulation of protein kinase

The second important regulatory function of diacylglycerol is stimulation of protein kinase C (see 7.7). Protein kinase C is a protein kinase occurring in almost all cells, and has a regulating effect on many reactions of the cell. Characteristic for protein kinase C is its stimulation by Ca, diacylglycerol and phosphatidyl serine. 

Signaling pathways starting from receptor tyrosine kinases trigger stimulation of protein kinase C by activating phosphohpase Cy. An activating signal may also be despatched in the direction of protein kinase C - via activation of phospholipase CP -from G-protein-coupled membrane receptors (see Fig. 6.4). 

Furthermore, the LPS signal transduction involves the activation of G proteins, of phospholipases C and D, the formation of diacyl-glycerol (DG) and inositol triphosphate (IP3). DG mediates the stimulation of protein kinase C (PKC) and IP3 induces an increase of cytosolic Ca++ The LPS signaling pathway also involves tyrosine kinases, constitutive nitric oxide (NO) synthase (cNOS), cGMP-dependent protein kinase, Ca channels, calmodulin and calmodulin kinase [27,28], as well as the MAP kinases [29] ERK1, ERK2 and p38 [23], The intracellular events in response to LPS are due to lipid A because they are inhibited by polymyxin B which is known to bind lipid A [27] and they are reproduced by lipids A [30,31]. 

Influence of ionophores and synthetic stimulators of protein kinase C... 

Rapid phosphorylation of the other detected phosphoproteins does occur but no definite roles have yet been ascribed to them. The 33 kDa protein may be the S6 ribosomal protein involved in the control of protein synthesis. The 57 kDa protein has been identified as the regulatory suhunit of the cyclic AMP-dependent protein kinase [44]. Of the other proteins the 76, 43 and 20 kDa may be connected with the microfilaments (43 kDa actin, 76 kDa myosin light chain kinase and 20 kDa myosin light chain) but this must be further investigated. These proteins may only play a permissive role in. steroidogenesis. The fact that the pattern of protein phosphorylation is very similar after stimulation of protein kinase C with phorbol esters supports this because the latter only marginally increase steroidogenesis [18]. 

The release of fatty acids from adipose tissue is regulated by the rate of hydrolysis of triacylglycerol and the rate of esterification of acyl-CoA with glycerol 3-phosphate. The rate of hydrolysis is stimulated by hormones that bind to cell-surface receptors and stimulate adenylate cyclase (which catalyzes the production of cAMP from ATP). Hormone-sensitive lipase (Sec. 13.4) can exist in two forms, one of which exhibits very low activity and a second which is phosphorylated and has high activity. Before hormonal stimulation of adenylate cyclase, the low-activity lipase predominates in the fat cell. Stimulation of protein kinase by an increase in cAMP concentration leads to phosphorylation of the low-activity lipase. An increase in the rate of hydrolysis of triacylglycerol and the release of fatty acids from the fat cell follows. This leads to a greater utilization of fatty acids by tissues such as heart, skeletal muscle, and liver. 

This chapter has emphasized the role of DNA mutations in the pathways leading to cancer. However, a number of chemicals are cancopromoting purely because of their ability to activate certain enzymes, where there exists no direct or indirect influence on DNA mutations. This functional class of compounds promotes or prevents cancer orJy when present on a chronic basis, i.e, at elevated levels every day for many years. The notion that bile salt metabolites may result in chronic stimulation of protein kinase C was discussed earlier. The related event of fiber decreasing exposure of the colon to bile salts was also outlined. The chronic exposure theory applies to the phorbol esters that are present in certain plants. People who drink tea made from the plant Croton flaveti tend to acquire esophageal cancer. This cancer results from chronic exposure to phorbol esters, which occurs in the leaf extract or in an oil prepared from the plant (croton oil). The phorbol esters enter the cell, bind to protein kinase C, and activate this enzyme. Activated protein kinase C, in turn, activates the MAP kinase cascade (Ueda et aL, 1996). The continual activation of protein kinase C, when combined with mutations in specific proto-oncogenes, may lead to cancer and sustain the cells in the cancerous state. 

By decreasing the activity of glycolytic enzymes competing for the same substrate in this case, by inactivating pyruvate kinase in the cytosol and pyruvate dehydrogenase in the mitochondria (via cAMP stimulation of protein kinase). 

In addition to activation by stretch, MAPK activity rises in response to pharmacological stimulation in contractile smooth muscle (Adam et al., 1995 Katoch and Moreland, 1995). Porcine carotid arteries stimulated with KCl, phorbol 12,13-dibutyrate (PDBu), or histamine demonstrate elevated levels of active MAPK. The activities of p42 APK g d p44MAPK jj-,. crease in tandem under all conditions tested that is, KCl, PDBu, and histamine do not preferentially activate one MAPK isoform over the other isoform. With KCl and histamine stimulation, MAPK activity reaches a maximum within several minutes. PDBu induces a more slowly developing increase in MAPK activity that is maintained for at least one hour. These data suggest that increases in intracellular free calcium, and/or the stimulation of protein kinase C, in vascular smooth muscle result in MAPK activation. Pharmacological increases in MAPK activity are in addition to the levels that result from the application of mechanical load. 

Angiotensin II, vasopressin, and endothelin inhibit K tp channels in cultured coronary artery smooth muscle cells (e.g., Miyoshi et al, 1992). Muscarinic receptor stimulation inhibited K jp channels in smooth muscle cells in urinary bladder through stimulation of protein kinase C (Bonev and Nelson, 1993b). Serotonin, phenylephrine, histamine, and neuropeptide Y also inhibit K jp currents in smooth muscle cells from mesenteric arteries through stimulation of protein kinase C (Bonev and Nelson, 1995). 

The c-GMP produced could activate a c-GMP dependent kinase which then might phosphorylate the transporter either directly or indirectly. Interestingly direct stimulation of protein kinase C by phorbol-12-myristate-13-acetate leads to a decrease in 5-HT uptake by a decrease in V ax-... 

The atypical protein kinase is ubiquitously expressed. It is considered atypical in that it is not activated by diacylglycerol or pre-treatment with phorbol esters. In addition, the protein does not undergo translocation from cytosol to membrane when activated. Phosphatidylinositol 3,4,5-triphosphate has been shown to result in a large stimulation of protein kinase t, (Nakanishi et al. 1993). 

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